Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS

The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field.     

A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts

Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac⁺ by prophage precise excision with a relatively high frequency (about 1×10⁻⁶). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced β-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.     

Encephalitozoonosis in pharmacologically immunosuppressed mice

Encephalitozoon cuniculi is a parasite that has been identified as a cause of opportunistic infections in immunocompromised individuals. This study was performed to evaluate E. cuniculi infection in pharmacologically immunosuppressed mice. Mice were immunosuppressed with cyclophosphamide (100mg/kg twice a week, IP) or cyclosporin (10mg/kg daily, IP) and inoculated with 10(7)E. cuniculi spores IP. The E. cuniculi spores were cultivated in MDCK cells. E. cuniculi identification was performed by light microscopy studies using Gram-Chromotrope, Hematoxylin-Eosin and Toluidine blue-fuchsin staining techniques, as well as by PCR at 15, 30 and 45 days post-inoculation (DPI). Cyclophosphamide-immunosuppressed mice have greatly reduced amounts of CD8(+), CD4(+) and CD3(+) T cells and CD19(+) B cells. The cells from these mice were analyzed by FACS and showed acute disseminated and fatal encephalitozoonosis. Mice treated with ciclosporin, which is both antiparasitic and immunosuppressive, have a milder, chronic, non-lethal infection and showed a significant reduction only in CD3(+) and CD4(+) T cell numbers. Our results support the role of CD8(+) T cells in controlling infection by E. cuniculi and show that preventive measures are essential for preventing this zoonosis in individuals undergoing chemotherapy for cancer or other immunosuppressive therapies.     

FtsZ dimerization in vivo

A hybrid assay, based on the properties of the lambda repressor, was developed to detect FtsZ dimerization in Escherichia coli in vivo. A gene fusion comprising the N-terminal end of the lambda cI repressor gene and the complete E. coli ftsZ gene was constructed. The fused protein resulted in a functional lambda repressor and was able to complement the thermosensitive mutant ftsZ84. Using the same strategy, a series of 10 novel mutants of FtsZ that are unable to dimerize was selected, and a deletion analysis of the protein was carried out. Characterization of these mutants allowed the identification of three separate FtsZ portions: the N-terminal of about 150 amino acids; the C-terminal of about 60 amino acids, which corresponds to the less conserved portion of the protein; and a central region of about 150 residues. Mutants belonging to this region would define the dimerization domain of FtsZ.     

Identification of Encephalitozoon and Enterocytozoon (microsporidia) spores in stool and urine samples obtained from free-living South American Coatis (Nasua nasua)

This study emphasizes the importance of free-living coatis as a potential source of microsporidian infection for humans living in large cities. We found 19 (31.7%) positive results among 60 fecal samples analyzed by PCR-based analysis and the Gram-Chromotrope staining technique (11.7% were positive for Encephalitozoon cuniculi, 6.7% for E. intestinalis, 6.7% for E. hellem, and 6.7% for Enterocytozoon bieneusi). Only 5 (8.4%) urine samples tested positive for E. cuniculi as assessed by the two techniques.     

Filamenting temperature-sensitive mutant Z inhibitors from Glycyrrhiza glabra and their inhibitory mode of action

FtsZ is an essential protein for bacterial cell division, and an attractive and underexploited novel antibacterial target protein. Screening of Indonesian plants revealed the inhibitory activity of the methanol extract of Glycyrrhiza glabra on the Bacillus subtilis FtsZ (BsFtsZ) GTPase, and further bioassay-guided fractionation of the active methanol extract led to the isolation of seven known polyketides (1–7). Among them, gancaonin I (1), glycyrin (3), and isolicoflavanol (5) exhibited anti-BsFtsZ GTPase activities, at levels comparable to that of the synthetic FtsZ inhibitor, Zantrin Z3. Enzymatic assays using a BsFtsZ Val307R mutant protein and in silico simulations suggested that 1, 3, and 5 bind to the cleft on BsFtsZ, as in the case of the previously reported uncompetitive FtsZ inhibitor, PC190723, and thereby display their significant anti-BsFtsZ inhibitory activities. Furthermore, 1 also showed significant inhibitory activity against B. subtilis, with a MIC value of 5 μM. The present study provides new insights into the naturally occurring B. subtilis growth inhibitors.     

Patterns of stress,disease and mortality in two prehistoric populations from North American

Archaeological data from the Dickson Mounds site was employed to reconstruct the cultural-ecological context of two prehistoric American Indian populations. These populations were identified as the Mississippian Acculturated Late Woodland (A.D. 1050–1200) and the Spoon River Focus of the Middle Mississippian (A.D. 1200–1300). The cultural-ecological data was then employed to interpret some of the biological parameters of the two skeletal populations.The cultural-ecological variables included: (a) subsistence technology; (b) population density and degree of sedentarism; and (c) the extent and intensity of contacts with surrounding social groups. The biological parameters included: (a) infectious disease; (b) micro-structural dental defects; and (c) mortality.The results of the analysis suggested that as alterations occurred in the cultural-ecological adaptations of the populations in the form of an increased reliance upon maize agriculture, an increase in population density and sedentarism, and an increase in social contacts; there occurred an increase in the frequency of infectious disease, micro-structural dental defects and mortality.     

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8⁺ T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID + B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-γ, TNF-α and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.     

Histological enamel indicator of childhood stress in prehistoric skeletal samples.

Although previous paleopathological studies have used disturbances in enamel formation as indicators of childhood stress, the full potential of this technique has not been realized. This paper presents a test case which demonstrates that the frequency of disturbed enamel formation (i.e., Wilson bands) is associated with other stress indicators (i.e., probability of dying and infectious lesions) in three prehistoric skeletal samples representing the Middle Woodland (10.3%), Mississippian Acculturated Late Woodland (21.4%), and the Middle Mississippian (40.0%). Additionally, the mean ages at death of individuals with at least one Wilson band are lower than those without bands.These results suggest that Wilson bands are an indicator of the relative proportion of individuals who are high susceptibles in prehistoric populations. The data also corroborate the hypothesis that the adoption of maize agriculture in the prehistoric American Midwest is associated with increased stress.